4 research outputs found
Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal
Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants
Triple Bioaffinity Mass Spectrometry Concept for Thyroid Transporter Ligands
For the analysis of thyroid transporter ligands, a triple
bioaffinity
mass spectrometry (BioMS) concept was developed, with the aim at three
different analytical objectives: rapid screening of any ligand, confirmation
of known ligands in accordance with legislative requirements, and
identification of emerging yet unknown ligands. These three purposes
share the same biorecognition element, recombinant thyroid transport
protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass
spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive
inhibition MS binding assay was developed with fast ultrahigh performance-liquid
chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS)
as the readout system. It uses the nonradioactive stable isotopic
thyroid hormone <sup>13</sup>C<sub>6</sub>-l-thyroxine as
the label of which the binding to rTTR is inhibited by any ligand
such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs).
To this end, rTTR is either used in solution or immobilized on paramagnetic
microbeads. The concentration-dependent inhibition of the label by
the natural thyroid hormone l-thyroxine (T4), as a model
analyte, is demonstrated in water at part-per-trillion and in urine
at part-per-billion level. For confirmation of identity of known ligands,
rTTR was used for bioaffinity purification for confirmation of naturally
present free T4 in urine. As a demonstrator for identification of
unknown ligands, the same rTTR was used again but in combination with
nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples
spiked with the model “unknown” EDCs triclosan and tetrabromobisphenol-A.
This study highlights the potential of BioMS using one affinity system,
both for rapid screening and for confirmation and identification of
known and unknown emerging thyroid EDCs
High-Throughput Bioaffinity Mass Spectrometry for Screening and Identification of Designer Anabolic Steroids in Dietary Supplements
A generic high-throughput bioaffinity
liquid chromatography-mass
spectrometry (BioMS) approach was developed and applied for the screening
and identification of known and unknown recombinant human sex hormone-binding
globulin (rhSHBG)-binding designer steroids in dietary supplements.
For screening, a semi-automated competitive inhibition binding assay
was combined with fast ultrahigh-performance-LC-electrospray ionization-triple-quadrupole-MS
(UPLC-QqQ-MS). 17β-Testosterone-D<sub>3</sub> was used as the
stable isotope label of which the binding to rhSHBG-coated paramagnetic
microbeads was inhibited by any other binding (designer) steroid.
The assay was performed in a 96-well plate and combined with the fast
LC-MS, 96 measurements could be performed within 4 h. The concentration-dependent
inhibition of the label by steroids in buffer and dietary supplements
was demonstrated. Following an adjusted bioaffinity isolation procedure,
suspect extracts were injected into a chip-UPLCÂ(NanoTile)-Q-time-of-flight-MS
system for full-scan accurate mass identification. Next to known steroids,
1-testosterone was identified in three of the supplements studied
and the designer steroid tetrahydrogestrinone was identified in a
spiked supplement. The generic steroid-binding assay can be used for
high-throughput screening of androgens, estrogens, and gestagens in
dietary supplements to fight doping. When combined with chip-UPLC-MS,
it is a powerful tool for early warning of unknown emerging rhSHBG
bioactive designer steroids in dietary supplements